Etude de deux enzymes paralogues MraY et WecA impliquées dans la biosynthèse de la paroi bactérienne, membres d’une famille de protéines membranaires Polyprényl-phosphate n-acétyl-hexosamine 1-phosphate transférases

A study of two paralogue enzymes MraY and WecA inovolved in the bacterial cell wall biosynthesis, members of a superfamily of membrane proteins polyprenyl-phosphate n-acetyl-hexosamine1-phosphate transferases

• Auteur / Autrice : Bayan Al-Dabbagh
• Direction : Ahmed Bouhss
• Type : Thèse de doctorat
• Discipline(s) : Sciences biologiques. Ingénierie des protéines
• Date : Soutenance en 2008
• Etablissement(s) : Paris 11
• Partenaire(s) de recherche : Autre partenaire : Université de Paris-Sud. Faculté des sciences d'Orsay (Essonne)

Mots clés

Mots clés contrôlés

Antibiotiques

Peptidoglycanes

Mots clés libres

Protéines membranaires

Résumé

L’enzyme mray catalyse la premiere etape membranaire de la biosynthese du peptidoglycane. Elle est essentielle à la viabilite de la bacterie, ce qui fait de cette enzyme une cible privilegiee pour la recherche de nouveaux antibiotiques. La topologie membranaire de mray a ete determinee. Elle est composee de 10 segments transmembranaires, 4 sequences periplasmiques, et 5 sequences cytoplasmiques contenant de nombreux residus invariants. Une partie de ce travail de these a concerne la cartographie du site actif de la translocase mray et l’etude de son mecanisme catalytique. Pour cela, dix-neuf proteines mutantes de mray de bacillus subtilis ont ete produites par mutagenese dirigee des residus invariants. Les plasmides portant ces mutations ont ete testes par complementation fonctionnelle in vivo. Quatorze proteines mutantes, qui n’assurent pas la complementation d’une souche mutante mray thermosensible, ont alors ete purifiees a homogeneite et leur analyse enzymatique a ete effectuee. Les etudes d’activite a differents ph suggerent l’implication du residu d98 dans la deprotonation de l’undecaprenyl-phosphate (c55-p) pendant la catalyse. L’activite enzymatique des proteines mutantes a differentes concentrations de mg2+ a ete determinee. Les resultats suggerent l’implication des residus d174, d177 et h45 dans la fixation du mg2+. L’etude du mecanisme catalytique de l’enzyme mray a ete entreprise. Les resultats de nos experiences supportent l’hypothese selon laquelle la formation du lipide i s’effectuerait selon un mecanisme catalytique en une etape, a savoir l’attaque directe d’un oxyanion du phosphate de l’undecaprenyl-phosphate sur le phosphate ß du substrat nucleotidique. Un autre volet de ce travail a ete consacre a l’etude de la transferase membranaire weca, paralogue de mray et membre de la meme famille d’enzymes, les polyprenyl-phosphate n-acetyl-hexosamine 1-phosphate transferases. Pour la premiere fois, nous avons reussi la surproduction, l’extraction des membranes et la purification a homogeneite de cette enzyme. Sa caracterisation biochimique a egalement ete effectuee. Une partie importante de cette these concerne l’etude et la recherche de nouveaux antibiotiques. Sur la base de la structure de la liposidomycine, un inhibiteur naturel de mray, de nouveaux composes ont ete synthetises. Les effets de ces derniers sur l’activite enzymatique de mray ont ete analyses. Une inhibition interessante a ete observee avec un de ces composes avec une ic50 de 0,58 mm. La derniere partie de cette these concerne l’etude de l’interaction entre les intermediaires lipidiques de la biosynthese du peptidoglycane et un nouveau lantibiotique produit par bacillus claucii, baptise claucine. Ce travail a ete realise par des approches de biophysique. Nous avons montre que les lipides i et ii sont des cibles de ce nouveau lantibiotique. En revanche, l’undecaprenyl-phosphate, l’udp-murnac-pentapeptide ou son analogue pyrophospho-murnac-pentapeptide n’interagissent pas avec la claucine.

The mray transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-n-acetylmuramoyl pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. Nineteen polar residues located in the five cytoplasmic segments of mray appeared as invariants in the sequences of mray orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the bacillus subtilis mray as a model. Fourteen residues were shown to be important for mray activity by an in vivo functional complementation assay using a constructed conditional mray mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of both the nucleotidic and lipidic substrates, but the kcat was drastically reduced in almost all cases. The important residues for activity appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the d98 residue which is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process. The effects of magnesium (required for mray activity) on the activity of the wild-type and mutant proteins were tested. Moreover, our results suggest the involvement of h45, d174 and d177 residues in the binding of the metal ion. We also studied the catalytic mechanism of mray. Based on our present results, we here propose a reaction proceeding via a single displacement mechanism. In this “one-step” mechanism, the oxyanion of the undecaprenyl-phosphate attacks the β phosphate of the nucleotidic substrate leading to the formation of lipid i and the liberation of ump. During this thesis, we were also interested in studying the weca transferase, an integral membrane protein, paralogue of mray. In this thesis and for the first time, the weca protein was overproduced and purified to near homogeneity in milligram quantities. Its kinetic parameters and biochemical characterization were determined. Another aspect of our work concerned the research of new antibacterial drugs. Indeed, mray, an essential integral membrane protein is a promising potential target to be exploited for new antibacterial discovery. Based on the structure of the liposidomycin, a natural inhibitor of mray, new compounds were synthesized. Their effects on the enzymatic activity of mray were analyzed. In the case of one compound, an important inhibition was observed with an ic50 of 0. 58 mm. In the last part of this manuscript we investigated the specificity of interaction of a new type a lantibiotic, termed claucin, isolated from bacillus claucii with the lipid precursors of the peptidoglycan biosynthesis. Our data show that these lipid intermediates are the targets of this new lantibiotic. However, no interaction was observed when undecaprenyl-phosphate, udp-murnac-pentapetide and its analogue the pyrophosphate-murnac-pentapeptide were tested.